American dog ticks along their expanding range edge in Ontario, Canada

The American dog tick, Dermacentor variabilis, is a tick of public and veterinary health importance in North America. Using passive tick surveillance data, we document distribution changes for the American dog tick in Ontario, Canada, from 2010 through 2018. Dermacentor variabilis submissions from the public were geocoded and aggregated—from large to small administrative geographies—by health region, public health unit (PHU) and Forward Sortation Area (FSA). PHU hot spots with high rates of D. variabilis submissions were (1) Brant County, Haldimand-Norfolk and Niagara Regional in the Central West region and (2) Lambton and Winsor-Essex County in the South West region. The number of established D. variabilis populations with ≥ 6 submissions per year increased significantly during the study at regional (PHUs: 22 to 31) and local (FSAs: 27 to 91) scales. The range of D. variabilis increased similarly to the positive control (Ixodes scapularis) during the study and in contrast to the static range of the negative control (Ixodes cookei). Submission hot spots were in warmer, low elevation areas with poorly drained soils, compared to the province’s low submission areas. Dermacentor variabilis is spreading in Ontario and continued research into their vector ecology is required to assess medicoveterinary health risks.


Materials and methods
Study location. Ontario has a population of approximately 14 million and is located in the Great Lakes region of North America. Most of southern Ontario experiences a moderate continental climate and lies within the Mixedwood Plains Ecozone (≈ 83,000 km 2 ); field and agricultural land comprise 59% of land cover in this ecozone (see citation for details of Ontario's ecozones) 22,23 . Oaks, maples, yellow birch, ashes, eastern hemlock, American beech, American elm, basswood, wild black cherry, hickories, eastern white pine, firs and spruce dominate forested areas of southern Ontario 22,23 . In successional habitats, common plants include staghorn sumac, highbush cranberry, clover, red osier dogwood, goldenrod and willow 22,23 .
Currently, 34 public health units (PHUs) administer public health services in Ontario; however, for this study, we performed analyses with the previous 35-PHU classification that were in existence during the time frame of the study (for PHU acronyms and full names, see Supplementary Table S1 Passive tick surveillance. We have described Ontario's passive tick surveillance previously 24 . Briefly, Public Health Ontario (PHO) morphologically identifies ticks submitted by the public through healthcare providers or PHU offices. This study spanned from 2010 to 2018 and only included ticks submitted from human hosts. Tick submission data include the submitter's postal code and travel history, along with tick submission date, stage, sex and number of ticks. The Forward Sortation Area (FSA) is the smallest geographic unit used in our analyses and is the first three characters of the six-character postal code of the submitter's residence. There are 513 FSAs in Ontario with a median area of 24.7 km 2 (interquartile range [IQR]: 8.7-151 km 2 ; range: 0.3-408,433 km 2 ), with geographically smaller FSAs in urban centers and larger FSAs in rural areas (see citation for further details on Ontario's FSAs) 25 . The median area of the 35 PHUs was 3,806 km 2 (interquartile range [IQR]: 2,036-8,988 km 2 ; range: 630-266,291 km 2 ). We used the submitter's FSA to aggregate data to the PHU level and then to the larger health region level. Ticks potentially acquired outside of Ontario were excluded from analyses (n = 389). EOH, KFL and LGL stopped accepting tick submissions at their PHU offices in 2014 and HDN stopped in 2018; however, healthcare providers could still submit ticks from patients in these PHUs (for provincial summary, we included a subset of PHUs that excludes PHUs that ceased submissions during the study period).
We used existing criteria to determine if D. variabilis was established: a PHU or FSA had an established population if the public submitted at least six ticks in a year from that PHU or FSA. This criterion is based on research focusing on I. scapularis and Ixodes pacificus in the United States of America (USA) and applied at the county-level geography; the criterion has also been applied to D. variabilis and Amblyomma americanum in the USA (lone star tick) [26][27][28][29] . We compared D. variabilis with a relatively newly-established tick (I. scapularis = positive control; a species demonstrated as having an expanding range) and a long-established, nidicolous tick (Ixodes cookei = negative control; a species with a relatively static range) 6,30,31 . We also use the term occurrence to describe a tick's distribution; occurrence is when the public submitted at least one tick from a PHU or FSA in a single year.
Mapping and statistical analyses. We calculated provincial, health region and PHU submission rates of D. variabilis per 100,000 population (denominator = 2018 population data) using population data and estimates from Statistics Canada via IntelliHEALTH Ontario (October 19, 2017). We created distribution maps using Esri ArcGIS v10.3 (Esri; Redlands, California, USA; 2014) for tick submission rates for health regions and PHUs. We did not map submissions by FSA, as the high variability in FSA size makes mapping at this level of geography problematic (e.g., most urban FSAs are too small to see on a map and large, rural FSAs would dominate the map). In addition, rate of submission at the FSA-level was out-of-scope for this work since population data for FSAs (including yearly and monthly projections) were not available.
We modelled D. variabilis submission counts using a Poisson regression and the number of submissions as the dependent variable, including year (linear), health region or PHU and their interaction term with year to determine if there are different trends over time by geography (reported as relative risk [RR], 95% confidence interval [CI]). The model also accounted for seasonality with sine and cosine with periods of pi/3 and pi/6 on the 12 months within a year. Population for each month, year and PHU/health region are included as an offset term in the model on the log scale. To examine the geospatial spread of the ticks over time, we modelled the number of FSAs with at least one submission as outcome using Poisson regression, accounting for seasonality (same periods as the model above) and time (in year) without an offset term. R v3. 6 were adult ticks, < 0.1% (n = 9) were nymphs and < 0.1% (n = 5) were mixed-stage submissions. For adult American dog tick submissions, 61.5% (10,717/17,414) were female, 36.4% (n = 6,334) were male and 1.9% (n = 337) were mixed-sex (i.e., sample contained male and female specimens). Submissions peaked during epidemiological week (epi week) 22 or approximately late May (Fig. 1).

Discussion
Dermacentor variabilis is a potential vector of several pathogens of public and/or veterinary health concern and, in Ontario, is expanding its range from key hotspots of relatively high abundance. Ontarians submitted D. variabilis from all health regions and PHUs of the province, with the highest submission rates from the Central West (BRN, HDN and NIA) and South West (LAM and WEC) regions. In addition, these hot spots experienced the highest multiple-tick submission rates, suggesting higher densities of ticks that potentially act as sources of expansion. While not a part of our analyses, submission hot spots were in the Mixedwood Plains Ecozone; specifically, in areas with higher temperatures (> 2,200 growing-degree-days above 5 °C) and lower elevation (< 225 m) 32 . The characterization of these high-submission areas is similar to studies showing that the American dog tick favours conditions associated with low elevation (e.g., higher humidity, higher temperature), favouring expansion and establishment 10,[33][34][35] . In neighbouring Michigan, USA, American dog ticks were more abundant along large bodies of water in low elevation areas of the state's southeast region and Northern Peninsula, contiguous with high submission areas of Ontario 36 . In southern Maine, USA, D. variabilis were more common in warmer areas at low elevation 31 . High-submission areas in Ontario have a high-percentage of poorly-drained, gleysolic soils (associated with wetlands or where wetlands once existed), consistent with reports of increased D. variabilis abundance in high-humidity habitats with damp soils 32,37,38 . There is an opportunity to model suitable habitat for D. variabilis in Ontario at a finer geographic scale (i.e., FSA), using passive and active surveillance data under current and future climate projections. Submission rates and counts for D. variabilis are increasing in Ontario; however, we did not confirm increasing relative abundance at the provincial level during the study. We received approximately 2,400 D. variabilis annually during this study (2013-2018), higher than previous passive surveillance work in Ontario.  2016) reported that D. variabilis is "locally abundant in southern Québec and Ontario" 2 . While passive surveillance was not able to detect increased D. variabilis relative abundance at the provincial level, abundance changes at the local level may well be determined in the future using active surveillance (e.g., tick dragging).
Undoubtedly, range expansion of D. variabilis was in progress for years to decades prior to our study, yet we demonstrate expansion in Ontario over a short period of 9 years and provide a baseline for future comparisons.  21,31,40 . Dermacentor variabilis range expansion indicates conditions are favourable for this tick across an area larger than they currently occur in Ontario (continued expansion), potentially increasing threats to public and veterinary health.
Relatively fast increases in D. variabilis submissions identified areas where the ticks are emerging or continuing to proliferate. The greatest increase in D. variabilis submissions over the study period were in the North East region, which was unexpected given the region consisted mostly of PHUs without established tick populations. Submissions increased annually by almost 50% in ALG, the PHU driving the rapid increase in the North East. The recent increase in D. variabilis in ALG (centered in and around Sault Ste. Marie) is likely the result of expansion of D. variabilis populations from the Upper Peninsula of Michigan, or possibly successful introduction and establishment of adventitious ticks from southern Ontario. We presume D. variabilis dispersal in agricultural southern Ontario occurs via host movement among patches of suitable habitat or stepping stones (e.g., isolated woodlots within cultivated land) 41 . The variability in the rate of increase of submissions by PHU may reflect variability in the size and proximity of these stepping stones (slower where stepping stones are widespread and small), in conjunction with variability in suitable climate and ecological requirements. Geographic expansion of D. variabilis in Ontario does not represent diffusion from a single historical population, rather a mosaic of hot spots expanding at variable rates toward areas with favourable habitat and climate.
While not specifically examined in this study, the range expansion of the American dog tick is likely due to a combination of climate change (e.g., higher annual temperatures and milder winters), changes in land use patterns (e.g., succession of abandoned agricultural land and forest fragmentation), changes in host distribution/ abundance (e.g., white-tailed deer, Odocoileus virginianus) and increased human travel (including dog travel). Increasing temperature is an important driver for the range expansion of D. variabilis and other ticks in North America, such as the blacklegged tick and the lone star tick 30,42 . Worldwide, other tick species are experiencing range expansions, such as I. ricinus in Northern Europe and Dermacentor reticulatus in Central Europe 43,44 . Dermacentor variabilis is hypothetically expanding along waterways found in high-submission areas of Ontario, similar to American dog tick expansion in Saskatchewan, Canada, along the South Saskatchewan River and Diefenbaker Lake 21 . For example, hosts travelling along waterways of the Grand River Watershed in Ontario's Central West region potentially aid the gradual dispersal of adult D. variabilis, especially host animals with relatively larger home ranges such as coyotes, red foxes (Vulpes vulpes) and white-tailed deer. In contrast to other www.nature.com/scientificreports/ ixodid ticks, American dog ticks rarely feed on birds, limiting long-distance and rapid dispersal 2 . Rapid and long-distance dispersal of D. variabilis is usually via anthropogenic means, whereby adventitious ticks travel on humans, companion animals or livestock. There is an opportunity to test hypotheses underpinning D. variabilis expansion in Ontario using active and passive surveillance, which can aid in predicting the location of potential emerging populations. Adult D. variabilis activity in Ontario peaked from late May through early June, similar to elsewhere in the northern portion of the tick's range; e.g., May (Pennsylvania), late May through early June (Maine, Nova Scotia) and June (Quebec) 31,38,45,46 . The life cycle of the American dog tick normally takes two years in northern latitudes, longer depending on local host availability and climate 2,47 . In warmer climates, D. variabilis has a one-year life cycle with longer activity periods and multiple adult cohorts with two (Kentucky, USA) or three (Georgia, USA) activity peaks during the spring and summer 48,49 . It will be interesting to see if there will be earlier-season shifts in D. variabilis activity in North America due to warming temperatures.
The public and veterinary health importance of American dog ticks in Ontario deserves attention, especially since we have identified population hot spots and expansion. Rocky Mountain spotted fever and tularemia are the primary tick-borne disease threats posed by American dog ticks in Ontario. Rocky Mountain spotted fever is not reportable to public health officials in Ontario; however, researchers in 1978 described a locally acquired case near Ottawa, the only documented case acquired in Ontario 50 . In a 2006 report, a dog from Ottawa, with no history of travel, was seropositive for R. rickettsii 51 . Tularemia is endemic throughout Ontario; however, human infection is usually the result of skinning infectious animals (e.g., common muskrats, Ondatra zibethicus; eastern cottontails, Sylvilagus floridanus), rather than tick-borne transmission 52 55,56 . We infer that the risk of D. variabilis-borne disease in Ontario is low; however, the epidemiological significance of the American dog tick in the province is underexplored.
There are several caveats to our study, which are associated with any surveillance program based on the passive submission of specimens. The location of tick acquisition is not always the same as the submitter's place of residence; although we excluded ticks where the submitter indicated that they were acquired outside of Ontario. We assumed that most tick exposures occurred near the submitter's home; therefore, we did not take into account possible human travel within Ontario. Increased awareness of the surveillance program potentially contributed to American dog ticks being submitted from a wider geographic area; however, the extent of this awareness and its impact on submissions is unknown. We also assumed tick submission behaviour did not vary among PHUs; though, the public in areas accustomed to this tick may not have sought identification. In addition, PHUs potentially did not submit all D. variabilis for identification, as they are relatively easy to identify. Our surveillance system did not include submissions from non-human hosts, meaning important hosts were missed (e.g., dogs); future work including collections from companion animals may elucidate further information on the distribution of American dog ticks in Ontario. We acknowledge that the 6-tick criterion for deeming a PHU or FSA as having an established population is a proxy until Ontario-and species-specific benchmarks are developed; this criterion likely overestimates the number of "true established populations" in the absence of confirmatory tick dragging. Further work is needed to determine if the 6-tick criterion is applicable at multiple scales. We should note that additional research is needed to quantify the extent that Ontario population growth (approximately 12.9 million in 2011 and 13.4 million in 2016) and loss of farm land (approximately 64.8 million hectares in 2011 and 64.2 million hectares in 2016) contributes to the spread of the American dog ticks in Ontario, especially at the FSA level 57,58 . The impact of discontinued passive surveillance meant it was not possible to assess spatiotemporal changes in D. variabilis populations in the Eastern region and we possibly missed areas of high submission rates or areas with rapidly expanding populations.